The present invention relates to methodologies and molecular targets for the prevention and treatment of microbial infection of a mammalian host through the disruption of the Candida albicans homologue of adenylate cyclase-associated protein (CAP1) gene. Preferably, these methods and molecular targets may be used in the prevention and treatment of microbial infection of mammalian hosts such as immunocompromised patients at risk for opportunistic fungal infections, organ transplant patients, cancer patients undergoing chemotherapy, burn patients, AIDS patients, or patients with diabetic ketoacidosis.
Whether pathogenic or opportunistic, microorganisms have evolved numerous mechanisms to facilitate their establishment and proliferation in mammalian hosts. During initial infection, the interaction of a microorganism with its mammalian host can include attachment or adhesion to the host cell surface, and invasion of host cells, for example. In certain instances, this interaction can be nonspecific. In others, such microbial interaction involves the specific binding of the microorganism to a particular receptor or receptor complex expressed on the host cell surface. In turn, the binding event can trigger changes in the microorganism and/or the mammalian host cell, leading to the progression of infection.
Candida is an ubiquitous yeast recognized as the causative agent of candidiasis (Candida mycosis). At least 90% of the disorders are caused by the species Candida albicans, which is an opportunistic yeast that elicits only mild superficial infections in normal individuals. However, destabilization of the host-parasite equilibrium upon inopportune loss or deficiencies in protective innate and immune deterrents favors overgrowth of the common gastrointestinal tract denizen and opportunistic pathogen, C. albicans. Acquired immunodeficiency syndrome (AIDS) or iatragenic immunosuppression are risk factors for oropharyngeal and esophageal candidiasis (Hood et al., 28 Clin. Infect. Dis. 587-96 (1999)). Thus, oropharyngeal and esophageal candidiasis are among the most frequent opportunistic fungal infections observed in human immunodeficiency virus positive (HIV+) and AIDS patients, occurring in the majority of patients. Candidal infections increase in severity and recur more frequently as the immunodeficiency progresses. The current status of the AIDS epidemic is one of increasing numbers of individuals infected and no cure. Many infected individuals may live for a long time with HIV in an essentially permanent immunocompromised state. Because of the loss of the cellular component of the immune system, AIDS patients are susceptible to invasion of submucosal tissue by C. albicans. In addition to HIV infected patients, oral candidiasis occurs in patients with leukemia or other cancers, as well as in patients with other underlying diseases. Prematurely-born infants are also at risk and may acquire mucosal infections causing permanent sequelae (Huang et al., 30 Scand. J. Infect. Dis. 137-42 (1998); Sood et al., 41 MYCOSES 417-9 (1998)). Candidiasis in denture wearers, or denture stomatitis, is the most common of all C. albicans associated diseases.
Although C. albicans is sensitive to antifungal drugs, treatment over long periods of time are required. At present, the treatment for invasive infections is based on relatively few antimycotics. Nystatin, ketoconazole, and amphotericin B are drugs which are used to treat oral and systemic Candida infections. However, orally administered nystatin is limited to treatment within the gut and is not applicable to systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these drugs may not be effective in such treatment unless combined with additional drugs. Amphotericin B has a relatively narrow therapeutic index and numerous undesirable side effects and toxicities occur even at therapeutic concentrations. While ketoconazole and other azole anti fungals exhibit significantly lower toxicity, their mechanism of action, inactivation of cytochrome P450 prosthetic group in certain enzymes (some of which are found in humans), precludes use in patients that are simultaneously receiving other drugs that are metabolized by the body""s cytochrome P450 enzymes. See, e.g., U.S. Pat. No. 5,863,762.
Other known antifungal agents include: polyene derivatives, such as amphotericin B (including lipid or liposomal formulations thereof) and the structurally related compounds nystatin and pimaricin; flucytosine (5-fluorocytosine); azole derivatives (including ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole, itraconazole, voriconazole [Pfizer] and SCH56592 [Schering-Plough]); allylamines-thiocarbamates (including tolnaftate, naftifine and terbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins (including MK-0991 [Merck]); nikkomycins; and bactericidal/permeability-increasing protein (BPI), described in U.S. Pat. Nos. 5,627,153; 5,858,974; 5,652,332; 5,763,567; and 5,733,872. Unfortunately, antimycotics cause serious, sometimes different, side effects, such as renal insufficiency, hypocalcemia and anemia, as well as unpleasant constitutional symptoms such as fever, shivering and low blood pressure.
The frequency of candidal infections may be a result of the prophylactic use of antibacterial drugs used in AIDS patients to minimize other opportunistic infections. Emergence of drug-resistant isolates and the limited selection of antifungal drugs point to the need for research aimed at identifying new anti-fungal targets (Terrell, 74 Mayo Clin. Proc. 78-100 (1999)). However, the pathogenesis is complex and is thought to involve multiple host factors that include loss of cell mediated immunity and altered phagocytic cell activity. High frequencies of nosocomial candidemia reflect the ability of C. albicans to translocate across the gastrointestinal tract, disrupting internal tissues in debilitated patients (Viscoli et al., 28 Clin. Infect. Dis. 1071-9 (1999)).
Thus far, studies have shown that development of candidiasis is a multi-stage process requiring sensing environmental conditions and transducing signals to regulate expression of appropriate genes at balanced levels in C. albicans. Filamentous growth of C. albicans includes not only pseudohyphal, elongated yeast-like forms described for Saccharomyces cerevisiae, but true hyphae as well. Compared to most pathogenic fungi, the morphological response of C. albicans to environmental conditions is rapid. Germ tubes are produced within one hour of placing cells in appropriate conditions. The mechanisms employed by C. albicans to achieve this apparently advantageous spectrum of growth morphologies and optimized metabolic activities are poorly understood.
A feature of C. albicans growth that is correlated with pathogenicity in the oral cavity is the ability to transform from budding to filament-extending growth. Filamentous forms adhere more readily to buccal epithelial cells than budding yeasts, and histologically are a prominent feature of invasion of the mucosa. In mucosal disease, filamentous forms, particularly true hyphae, invade the keratinized layer of differentiated, stratified squamous epithelium. True hyphae are septate, cylindrical structures with parallel sides that are formed by extension of germ tubes that emerge from yeasts in appropriate environmental conditions.
The relative contribution of yeast and filamentous forms to the pathogenesis of candidiasis is an unresolved issue. However, mutants that do not produce hyphae in vitro have reduced virulence in animal models (Ghannoum et al., 63 Infect. Immun. 4528-30 (1995); Lo et al., 90 CELL 939-49 (1997); Sobel et al., 44 Infect. Immun. 576-80 (1984)). Expression of hypha-specific virulence factors such as the hyphal wall protein (HWP1) adhesin gene (Staab et al., 283 Science 1535-38 (1999); Staab et al., 271 J. Biol. Chem. 6298-305 (1996)) and secreted aspartyl proteinase (SAP) genes (Schaller et al., 34 Mol. Microbiol. 169-80 (1999); Staib et al., 97 Proc . Natl. Acad. Sci. USA 6102-7 (2000)) are correlated with the virulence of hyphal forms. Research into the mechanisms that lead to the production of these virulence factors is important for developing strategies to interfere with candidiasis.
Thus, an alternative method to the prevention and treatment of candidiasis may be approached via disruption of molecular events that transform C. albicans to the pathogenic filamentous form. In many pathogenic fungi, interconversions between morphological growth forms, particularly between yeast growth and filamentous growth coincide with adaptation to a host environment followed by tissue destruction. Morphological interconversions in fungi are dependent upon signal transduction pathways including the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway (Bruno et al., 15 EMBO J. 5772-82 (1996); Gancedo, 25 FEMS Microbiol. Rev. 107-23 (2001); Kronstad et al., 170 Arch. Microbiol. 395-404 (1998); Lengeler et al., 64 Microbiol. Mol. Biol. Rev. 746-85 (2000)). For the plant pathogens Ustilago maydis and Magnaporthe grisea, cAMP signaling is important for the establishment of filamentous growth in the former and for formation of the infecting appressorium structure of the later (Kronstad et al., supra; Lengeler et al., supra).
Knowledge about how cAMP signaling mediates morphological interconversion is best understood for S. cerevisiae, a budding yeast that produces elongated pseudohyphal cells and forms filamentous colonies in the presence of limiting nitrogen (Gancedo, supra; Lengeler et al., supra). Pseudohyphal cells exhibit unipolar budding, do not separate and invade agar (Gimeno et al., 68 Cell 1077-90 (1992)). Recent experiments involving gene disruption and epistasis analyses have elucidated both upstream and downstream elements of the cAMP dependent pseudohyphal growth pathway in S. cerevisiae (Gancedo, supra; Kronstad et al., supra; Lengeler et al., supra). Adenylate cyclase is activated either through a receptor (Gpr1) that is coupled to a G protein (Gpa2) or by Ras2 (Gimeno et al., supra; Kxc3xcbler et al., 272 J. Biol. Chem. 20321-3 (1997); Lorenz and Heitman 16 EMBO J. 7008-18 (1997); Lorenz et al., 154 Genetics 609-22 (2000); Mxc3x6sch et al., 10 Mol. Biol. Cell. 1325-35 (1999); Toda et al., 40 Cell 27-36 (1985)). The subsequent activation of PKA then results in activation of the Flo8 transcription factor to produce a mucin-like protein, Flo11, that is localized to the cell surface and is required for pseudohyphal growth (Lambrechts et al., 93 Proc. Natl. Acad. Sci. USA 8419-24 (1996); Lo and Dranginis, 9 Mol. Biol. Cell. 161-71 (1998); Pan and Heitman, 19 Mol. Cell. Biol. 4874-87 (1999); Rupp et al., 18 EMBO J. 1257-69 (1999)). Although cross-talk between mitogen-activated protein kinase (MAPK) and cAMP signaling pathways is evident (Mxc3x6sch et al., supra), transcription factor targets important for filamentous growth appear not to be shared by the two pathways (Gancedo, supra; Lengeler et al., supra). Pseudohyphal defects caused by mutations in STE12 of the MAPK pathway and PHD1 are suppressed by constitutive activation of PKA through deletion of the regulatory subunit gene (BCY1) (Lo and Dranginis, supra).
Biochemical studies implicate cAMP increases in promoting bud-hypha transitions. Intracellular levels of cAMP increase and, under nutrient limitation, exogenous cAMP or dibutyryl cAMP (dbcAMP) increases the frequency of bud-hypha transitions (Chattaway et al., 123 J. Gen. Microbiol. 233-40 (1981); Niimi, 20 Fungal Genet. Biol. 79-83 (1996); Niimi et al., 142 J. Bacteriol. 1010-4 (1980); Zelada et al., 42 Cell. Mol. Biol. (Noisy-le-grand) 567-76 (1996)). Inhibitors of cAMP phosphodiesterase or cAMP-dependent protein kinase induce or block germ tube formation, respectively (Castilla et al., 10 Cell. Signal. 713-9 (1998), Chattaway et al., supra). However, genetic studies involving mutational analysis of genes that control cAMP levels and assessment of their roles in regulating bud-hypha transitions and filamentous growth have not been reported. Studies of the role of cAMP dependent signaling in morphogenesis will also bring to light common virulence pathways for distantly related fungal pathogens.
In S. cerevisiae, Ras activation of adenylate cyclase involves the adenylate cyclase protein (CAP, also known as Srv2p) (Fedor-Chaiken 61 Cell 329-40 (1990); Field et al., 61 Cell 319-27 (1990); Shima et al., 20 Mol. Cell. Biol. 26-33 (2000)). The CAP gene was identified in a genetic screen for mutants that suppressed defective growth of a strain carrying an inducible hyperactive RAS2va119 gene (Fedor-Chaiken, supra). The CAP gene was also isolated by screening a yeast cDNA expression library with antisera to a 70-KDa protein that co-purified with adenylate cyclase (Field et al., supra). CAP is required for normal budding morphology and growth rates in nutrient-rich media (Fedor-Chaiken, supra; Field et al., supra). Interestingly, the S. cerevisiae CAP gene has been shown to be involved in pseudohyphal differentiation using transposon mutagenesis to screen for mutant strains defective for filamentous growth (Mxc3x6sch et al., supra). CAPs of mice (Vojtek and Cooper, 105 J. Cell. Sci. 777-85 (1993)) and humans (Matviw et al., 12 Mol. Cell. Biol. 5033-40 (1992)) are 34% identical and 35% similar, respectively, to S. cerevisiae CAP showing that CAP genes are conserved throughout evolution. Although CAPs from different organisms have similar primary and secondary structures, the function of CAPs in developmental programs has diverged among fungi. CAP mutants of Schizosaccharomyces pombe but not S. cerevisiae conjugate and sporulate in inappropriate conditions (Kawamukai et al., 3 Mol. Biol. Cell. 167-80 (1992)).
Modulation of adenylate cyclase activity by CAP in S. cerevisiae (Field et al., supra; Yu et al., 274 J. Biol. Chem. 19985-91 (1999)) suggests that the CAP gene of C. albicans might affect intracellular cAMP levels, allowing assessment of the role of cAMP in the filamentous growth and virulence of C. albicans. In the present invention, the C. albicans CAP1 gene was cloned and its identity was established by sequence similarities to CAP gene products of other organisms, by the reduction in cAMP levels in cap1/cap1 mutants and by the ability of exogenous cAMP or dbcAMP to promote bud-hypha transitions and filamentous growth in cap1/cap1 mutants. cap1/cap1 mutants were unconditionally deficient in forming bud-hypha transitions and filamentous growth in rich and minimal, liquid and agar-based culture media, as well as in serum and saliva. cap1/cap1 mutants also showed reduced virulence in a systemic model of candidiasis. The present invention is the first to describe genetic evidence showing that cAMP promotes true hyphae formation in C. albicans. The present invention also describes interference with CAP1 function, which has potential for providing novel strategies for interfering with candidiasis.
By defining the molecular events leading to the expression of a morphogenically important gene, and through the identification of new genes that are co-regulated with CAP1, the present invention has strong potential for identifying new and novel ways to interfere with candidiasis. The long term medical benefits of the present invention may be the development of alternative or adjunctive therapies based on new knowledge about expression of CAP1 genes in C. albicans. Accordingly, an objective of the present invention includes identifying and characterizing the 5xe2x80x2 and 3xe2x80x2 sequences flanking the CAP1 gene.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. The detailed description and the specific examples, however, indicate only preferred embodiments of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The present invention relates to a method for disrupting the C. albicans homologue of adenylate cyclase-associated protein (CAP1) gene, which results in the interference of morphogenic transitions of the fungus. In a specific embodiment, disruption of the C. albicans CAP1 gene prevents the expression of the polypeptide capable of increasing cAMP levels which in turn stimulates bud/hypha transition. This particular embodiment of the present invention is accomplished through the construction of a cap1/cap1 mutant.
Another aspect of the invention is a purified polypeptide comprising the amino acid sequence of SEQ. ID. NO. 1, wherein said polypeptide C. albicans Cap1 is the gene product of the CAP1 gene (SEQ. ID. NO. 2). Another aspect is an isolated DNA molecule encoding the polypeptide having the amino acid sequence of SEQ. ID. NO. 1, and an isolated DNA molecule comprising the nucleotide sequence SEQ. ID. NO. 2 encoding the polypeptide of SEQ. ID. NO. 1. A further aspect is a nucleic acid capable of hybridizing under high stringency conditions to the DNA molecule of an isolated DNA molecule comprising the nucleotide sequence SEQ. ID. NO. 2 encoding the polypeptide of SEQ. ID. NO. 1.
An additional aspect of the invention is a microarray comprising at least one nucleotide sequence or fragment thereof, of the CAP1 gene (SEQ. ID. NO. 2). A further aspect is a method for detecting the expression of a protein capable of stimulating increases in cAMP levels in a microorganism, using microarrays and genome-wide expression. In a preferred embodiment, the microorganism is a bacteria or yeast, and more preferably C. albicans. 
In yet a further embodiment of the present invention, the patients may be immunocompromised and at risk for opportunistic fungal infections. In particular the patient may be, but is not limited to, an organ transplant recipient, a cancer patient undergoing chemotherapy, a burn patient, an AIDS patients, or a patient with diabetic ketoacidosis.
In a final embodiment, the present invention provides a methodology for characterizing genes under the control of Cap1 in a fungus. This embodiment is accomplished by creating a genomic library isolated from a fungus, specifically C. albicans, screening the genomic library with probes for genes identified by genome-wide expression profiling, and isolating and sequencing the resultant clones.